Research progress of immunodeficient animal models using gene modification techniques / 中国实验动物学报

The established immunodeficient animal models could be used as valuable resource for mechanism re-search of related disease in humans, drug discovery and development, translational research and stem cell research.How-ever, it is difficult and low-efficient to establish the genetic modified animal models using traditional technologies.The re-ports for immunodeficient animal models are few in middle-size and large animals.Recently, several effective gene-targeting tools, including ZFNs, TALENs, CRISPR/Cas9, develop quickly and provide technology basis for the establishment of im-munodeficient animal models.In this paper, the technology principles and research progresses of ZFNs, TALENs, CRISPR/Cas9 are introduced.The significant progresses of these emerging technologies achieved in immunodeficient ani-mal models are also elaborated, including KO Rag1/Rag2 rabbit, KO Rag1/Rag2 pig, KO IL2rg pig, KO Ppar-g/Rag1 monkey, and so on.In addition to being models for researching SCID-related diseases in humans, and evaluating the effica-cy and safety of stem-cell engraftment, these models may be also useful to develop surgical procedures for placement of grafts before clinical trials in humans, to produce humanized animals and bridge the gap between laboratory animal and medicical research.The immunodeficient animal models described here represent a step toward the comprehensive evalua-tion of preclinical cellular regenerative strategies.

Molecular cloning, sequence characterization and mRNA tissue expression analysis of TDRP1 gene from the Banna minipig inbred line (BMI) / 中国实验动物学报

Objective To get TDRP1 gene of sterile and fertile boar of the Banna minipig inbred line (BMI), predict its function by bioinformatics analysis, and detect its expression patterns in the fertile boar.Methods Based on the NM_001198925 sequence, we designed specific primers and amplified BMI TDRP1 using RT-PCR method for sequen-cing and bioinformatics analysis.Meanwhile, the expression of TDRP1 in 17 organ tissues ( heart, liver, spleen, lung, kidney, thymus, lymph nodes, skin, duodenum, stomach, cerebrum, cerebellum, testis, epididymis, seminal vesicle, prostate, and bulbourethral gland) of fertile BMI boar and in the testis of sterile and fertile BMI boars was analyzed by semi-quantitative RT-PCR.Results The experiment obtained 680 bp cDNA sequence ( GenBank accession number:KJ186786) of BMI TDRP1, which encodes a protein of 186 amino acids with a predicted molecular weight (Mw) of 20.49 kDa and isoelectric point (pI) 5.86, and no signal peptide.It was a nuclear protein with a probability of 94.1%and had a leucine-rich nuclear export signals.Homology analysis of protein sequences revealed that BMI TDRP1 showed high identi-ty with that of humans, macaca mulatta, mouse and rat.The RT-PCR analysis showed that TDRP1 had a similar expression in the testes of sterile and fertile BMI boars.It was highly abundant in the seminal vesicle and prostate, moderately ex-pressed in cerebellum and testis and weakly expressed in cerebrum and kidney, while undetected in other 11 organ tissues. Conclusions We have cloned TDRP1 complete coding sequence, and found 2 SNPs,showing no difference in sequences and the testis mRNA expression levels between the fertile and sterile BMI boars.The multi-tissue transcription profile shows different expression levels in different organ tissues, being high in the seminal vesicle and prostate.The results of this study provide a foundation for further insight into the role of this gene in spermatogenesis.

Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector / 生物医学工程学杂志

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

Variation of host cell tropism of porcine endogenous retroviruses expressed in chinese Banna minipig inbred.

A serious donor-organ shortage urges the use of animal donors to treat a wide appropriate variety of major health problems including organ failure and diabetes. However, the promise of clinical xenotransplantation is offset at the present time by the potential of a public health risk due to the cross-species transmission of pathogens from animal donors to human patients. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern. In this study, cell tropism of PERV was tested by in vitro infection of human primary cells and cell lines. Coculture of PERV supernatant derived from PK15 with human primary cells and cell lines resulted in the transfer and expression of PERV-specific sequences and the establishment of a productive infection. In the detection of tropism variation of PERV in pigs, 293 cells were cocultured with mitogenic-activated and lethally irradiated PBMC from 12 Banna minipig inbred (BMI). The results were that six coculture groups were PERV-positive. However, infectious virus was not detected when activated PBMC from the other 7 pigs were cocultivated with human cells known to be permissive for PERV, which indicated a tropism variation among the tested individuals. All these findings demonstrate that the presence of endogenous viruses in source animals needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

Antigen measurement and biomechanical characteristics of the inbred-line Banna mini-pig acellular bone matrix / 生物医学工程学杂志

In this study, we prepared the acellular bone matrix of the inbred-line Banna mini-pig by using tissue engineering method and evaluated its possible application in bone tissue engineering. Histological analysis, xenoantigen expression and biomechanical measurement were performed on the matrix. HE staining and scanning electron microscopy showed the cellular components were almost removed. Immunohischemical result demonstrated that the xenoantigen, alpha-gal,was also eliminated. There was no statistically significant difference between the acellular bone matrix group and control group. The acellular bone matrix can provide appropriate space structure and strength for grafts. In conclusion, our data suggest that acellular bone matrix is a new kind of ideal bone scaffold material.

Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with some type C retroviruses.

PCR amplification of proviral DNA extracted from peripheral blood lymphocytes of three Chinese pigs (Banna minipig inbreed (BMI), Wu-Zhi-Shan pig (WZSP) and Neijiang pig (NJP)), using primers corresponding to highly conserved regions of reverse transcriptase (RT) of pol gene and nucleocapsid sequence of gag gene. PCR products were then extracted and cloned into pGEM-T vector. Phylogenetic analysis of the nucleotide sequences of PERV-BMI, PERV-WZSP and PERV-WZSP revealed that they were of retroviral origin. Phylogenetic trees were constructed from the translated amino acids of PERVs and other type C retrovirus, as well as lentivirus of GenBank. The research demonstrated that PERVs of Chinese pigs and other PERVs were closely related to other pathogenic type C retroviruses. From the gag analysis, a novel subgroup of PERV was identified and this novel sequence described in this report would allow such investigation to be actively pursued.

The quantity analysis of reverse transcriptase in porcine endogenous retrovirus expressed in Banna minipig inbred / 生物医学工程学杂志

Quantitative RT(reverse transcriptase) assay was established to detect the reverse transcriptase in plasma of thirty-four Chinese Banna minipig inbred in this work. The protocol was given in the RT kit (Roche), using HIV-1 as the positive control of the kit and supernatant of PK-15 as the PERV positive control respectively. The results show that positive reverse transcriptase reaction can be detected in the plasma of the pigs, but the levels are much lower than that of HIV-1 and lower than that of PERV in supernatant of PK-15.